The combination of Eicosapentaenoic acid (EPA) and Docosahexaenoic acid (DHA) supplementation, two main types of Long-chain n-3 polyunsaturated fatty acids (LCn-3 PUFAs) found in fish oil, has previously been shown to increase protein anabolism in both young animal models (Gingras et al, 2007) and elderly humans (Smith et al, 2011) via the mTOR-p70s6k pathway. Individually EPA has been reported to reduce the effect of proteolytic factor (PIF) on protein degradation in C2C12 (Smith et al, 1999) and has also been found to attenuate the deleterious effects of TNF-alpha during skeletal muscle C2C12 differentiation (Magee et al, 2007). However, research into the distinct contribution of EPA and DHA to protein anabolism, are at present, limited. The present study aims to clarify the distinct effects of EPA, DHA and a mixture of EPA and DHA on anabolic signaling pathways in C2C12 myocytes. On differentiation day 4, C2C12 cells were incubated with differentiation medium containing either EPA (50µM), DHA (50µM), a mixture of EPA (50µM) and DHA (50µM) or control for 24 h. Cells were serum starved for 4 h, followed by amino acid deprivation period for 1 h and then stimulated with 2mM L-Leucine for 30 min. Phosphorylated and total amounts of Akt[Ser473], mTOR[Ser2448], 4EBP1[Thr37/46], p70s6k[Thr389] and rps6[Ser235/236] were measured by western blotting. Data were analysed by one-way ANOVA and independent t-tests. Data are expressed as mean ± S.E.M. EPA, DHA, and a heterogeneous mixture of EPA and DHA produced a similar increase (P<0.05) in p70s6k[Thr389] phosphorylation, a key protein in the initiation step of protein translation, compared to control condition. This increase was 14.8 ± 5.3 % for EPA, 24.6 ± 8.2 % for DHA, and 27.9 ± 11.2 % for a mixture of EPA and DHA with no change in phosphorylation of Akt[Ser473] and mTOR[Ser2448]. The phosphorylation of 4EBP1[Thr37/46] was increased to 84.3 ± 39.4 % from the control condition (P=0.04) in response to EPA stimulation but not in that of the other treatments. This study has demonstrated that EPA, DHA and a mixture of EPA and DHA can stimulate p70s6k activation. Interestingly, only EPA can activate the phosphorylation of 4EBP1 in C2C12 cells.